PIT-1 ACTIVATION OF THE GROWTH HORMONE CHROMATIN LOCUS

描述（由申请人提供） 这项申请的目的是大大扩大和加强在分子生物学领域的候选人的培训。这将在一个明确的指导计划下完成，通过扩大正在进行的小鼠转基因研究的范围，远程基因激活（目标I），并通过获得一套全新的生物化学和结构生物学工具，需要制定和执行一套补充的体外研究（目标II）。所提出的实验集中于人生长激素（hGH）染色质基因座的激活。生长激素基因已成为研究组织特异性和发育调控基因表达的范例。hGH基因簇具有特别的实用性，因为它也可以作为长距离基因激活的信息模型。一组远端调控元件，包括基因座控制区（LCR），对垂体和胎盘中hGH基因簇的表达施加主要调控控制。HGH LCR决定簇长距离激活其靶基因，并以发育有序和组织特异性的方式。详细了解hGH LCR的结构、功能和作用机制对于了解hGH基因调控和扩展可用的基因控制机制模型至关重要。一套具体的科学目标将在指导环境中解决，这将扩大候选人的专业能力和科学技能。目的我将扩大正在进行的hGH LCR活性的表征，通过开发一套新的转基因小鼠系，以评估是否需要在HSI的LCR的决定簇维持hGH基因在转录活性状态，一旦它被激活，并确定是否在HSI的Pit-1结合位点可以自主靶向组蛋白乙酰化。Aim II的体外研究将检测纯化的重组Pit-1与重组核小体中LCR的Pit-1元件结合的能力，随后将CBP/p300组蛋白乙酰化因子招募到Pit-1：核小体复合物中，并激活重组染色质模板的转录。这些目标将在两名致力于候选人培训的个人和各自领域的专家的指导下进行。申请的最终目标是促进对基因调控的理解，并使候选人能够在学术机构承担独立的研究项目。

DESCRIPTION (provided by applicant) The objective of this application is to substantially broaden and strengthen the training of the candidate in the field of molecular biology. This will be accomplished under a defined mentoring program by extending the scope of ongoing mouse transgenic studies of long-range gene activation (Aim I) and by acquiring an entirely new set of biochemistry and structural biology tools required to formulate and execute a complementary set of investigations in vitro (Aim II). The experiments that are proposed focus on activation of the human Growth Hormone (hGH) chromatin locus. The GH gene has served as a paradigm for the study of tissue-specific and developmentally-regulated gene expression. The hGH gene cluster is of particular utility as it also serves as an informative model for long-range gene activation. A set of distal regulatory elements, comprising a locus control region (LCR), exerts major regulatory control over expression of the hGH gene cluster in both the pituitary and placenta. HGH LCR determinants activate their target genes over long distances and in a developmentally ordered and tissue-specific manner. A detailed understanding of hGH LCR structure, function, and mechanisms of action is of central importance to understanding of hGH gene regulation and to expanding the available mechanistic models of gene control. A set of specific scientific goals will be addressed within a mentoring environment that will expand the candidate's professional abilities and scientific skills. Aim I will expand ongoing characterization of hGH LCR activities by developing a novel set of transgenic mouse lines to assess whether determinants at HSI of the LCR are required to maintain the hGH gene in a transcriptionally active state once it is activated, and to determine whether the Pit-1 binding sites at HSI can autonomously target histone acetylation. The in vitro studies of Aim II will test the ability of purified recombinant Pit-1 to bind to Pit-1 elements of the LCR in reconstituted nucleosomes, to subsequently recruit CBP/p300 histone acetylation factors to the Pit-1: nucleosome complexes, and to activate transcription from a reconstituted chromatin template. These Aims will be carried out under the mentorship of two individuals dedicated to the candidate's training and expert in their respective fields. The ultimate goal of the application is to advance the understanding of gene regulation and to position the candidate to assume an independent research program at an academic institution.